The key to future fluorescence sensing lies in the use of new methods for fluorescence modulation, beyond what is currently available. DNA-templated silver nanoclusters (DNA/AgNCs) offer such an opportunity as their fluorescence emission color can be tuned by their DNA hosts, not via the traditional modulation scheme such as FRET. The resulting complexes, called NanoCluster Beacons (NCBs), are a unique class of activatable, multicolor molecular probes that have already found applications in diverse fields. However, it is unclear what sequence features of the activator strands ultimately control the fluorescence activation “patterns” of various NCBs. Here we demonstrate a high-throughput selection method, based on an Illumina sequencing chip, that allows us to rapidly characterize fluorescence activation of 10⁴-10⁵ NCBs. We not only discover new NCBs that are brighter than the known best but also identify critical nucleobases (positions 7-12 in the activator, termed the critical zone) that are key to stabilizing bright NCBs. Furthermore, a machine learning model based on sequence motifs is proposed to accelerate the designing process of NCBs with different fluorescence characteristics. Lastly, we design and study “polar opposite twin” NCBs (POT-NCBs), which is a pair of NCBs that differ only by a single nucleotide in their activators but have drastically distinct activation intensities or colors. We demonstrate the fluorescence enhancement difference of a yellow POT and a red POT could be as large as 31-fold and 8-fold, respectively. Based on the findings of the critical zone for bright NCBs and the hotspots for generating POT-NCBs we propose a “zipper bag model” that explains how the POT-NCB hotspots influence on the NCB synthesis yield. We anticipate new NCB-based sensors and new fluorescence barcodes will soon be developed based on the design strategy that we layout in this work.

Yuan-I Chen

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